Voltage-gated sodium channels (VGSCs) are found in all excitable cells. In neuronal cells of the central nervous system (CNS) and peripheral nervous system (PNS), sodium channels are primarily responsible for generating the rapid upstroke of the action potential. In this manner, sodium channels are essential to the initiation and propagation of electrical signals in the nervous system. Proper function of sodium channels is therefore necessary for normal function of the neuron. Consequently, aberrant sodium channel function is thought to underlie a variety of medical disorders. See Hubner et al., Hum. Mol. Genet. 11:2435-2445 (2002) for a general review of inherited ion channel disorders, including epilepsy (Yogeeswari et al., Curr. Drug Target 5:589-602 (2004)), arrhythmia (Noble, Proc. Natl. Acad. Sci. USA 99:5755-5756 (2002)), myotonia (Cannon, Kidney Int. 57:772-779 (2000)), and pain (Wood et al., J. Neurobiol. 61:55-71 (2004)).
VGSCs are composed of one α-subunit, which forms the core of the channel and is responsible for voltage-dependent gating and ion permeation, and several auxiliary β-subunits (see e.g., Chahine et al., CNS & Neurolog. Disorders-Drug Targets 7:144-158 (2008) and Kyle and Ilyin, J. Med. Chem. 50:2583-2588 (2007)). The α-subunits are large proteins composed of four homologous domains. Each domain contains six α-helical transmembrane spanning segments. There are currently nine (9) known members of the family of VGSCs α-subunits, including SCNx, SCNAx, and Navx.x (see Table 1, below). The VGSC family has been phylogenetically divided into two subfamilies Nav1.x (all but SCN6A) and Nav2.x (SCN6A). The Nav1.x subfamily can be functionally divided into two subgroups, those which are sensitive to blocking by tetrodotoxin (TTX-sensitive or TTX-s) and those which are resistant to blocking by tetrodotoxin (TTX-resistant or TTX-r).
TABLE 1Voltage-Gated Sodium Channel Gene FamilyGeneTissueTTX IC50DiseaseTypeSymbolDistribution(nM)AssociationIndicationsNav1.1SCN1ACNS/PNS10EpilepsyPain, seizures,neurodegenerationNav1.2SCN2ACNS10EpilepsyEpilepsy,neurodegenerationNav1.3SCN3ACNS15—PainNav1.4SCN4ASkeletal muscle25MyotoniaMyotoniaNav1.5SCN5AHeart muscle2,000ArrhythmiaArrhythmiaNav1.6SCN8ACNS/PNS6—Pain, movementdisordersNav1.7SCN9APNS25ErythermalgiaPainNav1.8SCN10APNS50,000—PainNav1.9SCN11APNS1,000—Pain
There are three members of the subgroup of TTX-resistant sodium channels. The SCN5A gene product (Nav1.5) is almost exclusively expressed in cardiac tissue and has been shown to underlie a variety of cardiac arrhythmias and other conduction disorders (Liu et al., Am. J. Pharmacogenomics 3:173-179 (2003)). The remaining TTX-resistant sodium channels, Nav1.8 (SCN10A, PN3, SNS) and Nav1.9 (SCN11A, NaN, SNS2) are expressed in the peripheral nervous system and show preferential expression in primary nociceptive neurons. Aberrant expression of Nav1.8 has been found in the CNS of human multiple sclerosis (MS) patients and also in a rodent model of MS (Black et al., Proc. Natl. Acad. Sci. USA 97:11598-115602 (2000)). Evidence for involvement in nociception is both associative (preferential expression in nociceptive neurons) and direct (genetic knockout). Nav1.8-null mice exhibited typical nociceptive behavior in response to acute noxious stimulation but had significant deficits in referred pain and hyperalgesia (Laird et al., J. Neurosci. 22:8352-8356 (2002)).
The SCN2A gene product (Nav1.2) is normally expressed along premyelinated and myelinated axons at different stages of maturation and is also expressed in a subset of demyelinated axons (Boiko et al., Neuron 30:91-104 (2001)). The Nav1.2 may be a basis for the firing of action potentials, even with depolarization (Rush et al., J. Physiol. 3:803-815 (2005)). Nav1.2 channels expressed in neurons are therapeutic targets in seizure, stroke, and pain.
The Nav1.7 (PN1, SCN9A) VGSC is sensitive to blocking by tetrodotoxin and is preferentially expressed in peripheral sympathetic and sensory neurons. The SCN9A gene has been cloned from a number of species, including human, rat, and rabbit and shows about 90% amino acid identity between the human and rat genes (Toledo-Aral et al., Proc. Natl. Acad. Sci. USA 94:1527-1532 (1997)).
An increasing body of evidence suggests that Nav1.7 sodium channel may play a key role in various pain states, including acute, inflammatory and/or neuropathic pain. For Example, Nav1.7 sodium channel may play a role in inflammatory dental pain (Beneng et al., BMC Neuroscience 11:1-7 (2010)). Deletion of the SCN9A gene in nociceptive neurons of mice led to an increase in mechanical and thermal pain thresholds and reduction or abolition of inflammatory pain responses (Nassar et al., Proc. Natl. Acad. Sci. USA 101:12706-12711 (2004)).
Sodium channel-blocking agents have been reported to be effective in the treatment of various disease states, and have found particular use as local anesthetics, e.g., lidocaine and bupivacaine, and in the treatment of cardiac arrhythmias, e.g., propafenone and amiodarone, and epilepsy, e.g., lamotrigine, phenytoin and carbamazepine (see Clare et al., Drug Discovery Today 5:506-510 (2000); Lai et al., Annu. Rev. Pharmacol. Toxicol. 44:371-397 (2004); Anger et al., J. Med. Chem. 44:115-137 (2001), and Catterall, Trends Pharmacol. Sci. 8:57-65 (1987)). Each of these agents is believed to act by interfering with the rapid influx of sodium ions.
It has also been reported that sodium channel-blocking agents may be useful in the treatment of pain, including acute, chronic, inflammatory, neuropathic, and other types of pain such as rectal, ocular, and submandibular pain typically associated with paroxysmal extreme pain disorder (see, e.g., Kyle and Ilyin, J. Med. Chem. 50:2583-2588 (2007); Wood et al., J. Neurobiol. 61:55-71 (2004); Baker et al., TRENDS in Pharmacol. Sci. 22:27-31 (2001); and Lai et al., Current Opinion in Neurobiol. 13:291-297 (2003)).
It has further been reported that sodium channel-blocking agents may be useful in the treatment of neurological disorders such as epilepsy, seizures, epilepsy with febrile seizures, epilepsy with benign familial neonatal infantile seizures, inherited pain disorders, e.g., primary erythermalgia and paroxysmal extreme pain disorder, familial hemiplegic migraine, and movement disorder; and the treatment of other psychiatric disorders such as autism, cerebeller atrophy, ataxia, and mental retardation (see, e.g., Chahine et al., CNS & Neurological Disorders-Drug Targets 7:144-158 (2008) and Meisler and Kearney, J. Clin. Invest. 115:2010-2017 (2005)). In addition to the above-mentioned clinical uses, carbamazepine, lidocaine and phenytoin are used to treat neuropathic pain, such as from trigeminal neuralgia, diabetic neuropathy and other forms of nerve damage (Taylor and Meldrum, Trends Pharmacol. Sci. 16:309-316 (1995)). Furthermore, based on a number of similarities between chronic pain and tinnitus (Moller, Am. J. Otol. 18:577-585 (1997); Tonndorf, Hear. Res. 28:271-275 (1987)), it has been proposed that tinnitus should be viewed as a form of chronic pain sensation (Simpson et al., Tip. 20:12-18 (1999)). Indeed, lidocaine and carbamazepine have been shown to be efficacious in treating tinnitus (Majumdar et al., Clin. Otolaryngol. 8:175-180 (1983); Donaldson, Laryngol. Otol. 95:947-951 (1981)).
The polypeptide toxins from the tarantula Thrixopelma pruriens (protoxins) are members of the inhibitory cysteine-knot family of protein toxins, which contain 30 to 35 amino acid residues and three disulfide bridges. Protoxin I (ProTx I) and Protoxin II (ProTx II) are T. pruriens peptide toxins that inhibit activation of sodium channels (Middleton et al., Biochemistry 41:14734-14747 (2002)). ProTx I and ProTx II act as gating modifiers that prevent channel activation via a voltage sensor-trapping mechanism (Edgerton et al., Toxicon 52:489-500 (2008); Priest et al., Toxicon 49:194-201 (2007)). ProTx II inhibits Nav1.7 sodium channels (see Schmalhofer et al., Molecular Pharm. 74: 1476-1481 (2008)).
WO 2012/004664 A2 discloses the analogs of sodium channel peptide toxin, and the pharmaceutically acceptable salts, prodrugs and solvates thereof. The analogs are useful as blockers of sodium (Na+) channels, and particularly Nav1.7 channels. The entirety of WO 2012/004664 A2 is incorporated by reference herein.
WO 2012/004664 A2 discloses the following natural toxins and their sodium channel blocking properties:
TABLE 2Natural Toxins Sodium Channel AssaysSEQIDIC50 Nav1.7IC50 Nav1.2Sequence*NO:Name(nm)(nm)YCQKWMWTCDSERKCCEGMVCR1ProTx1   105 ± 20LWCKKKLWII YCQKWMWTCDSARKCCEGLVCR2PaTx I  423 ± 1105000LWCKKII YCQKWMWTCDSERKCCEGYVCE3JzTx1,527 ± 13073,939 ± 14,440LWCKYNLXII YCQKWLWTCDSERKCCEDMVCR4GsAF I   249 ± 20   255 ± 43LWCKKRL YCQKWMWTCDSKRACCEGLRCK5JzTx V    14 ± 10   157 ± 20LWCRKII YCQKWMWTCDEERKCCEGLVCR6VsTx9,261 ± 2,21042,409 ± 10,010LWCKKKIEEGII YCQKWMWTCDEERKCCEGLVCR7GsAF   70 ± 10   410 ± 20LWCKKKIEWII YCQKWMWTCDSKRKCCEDMVCQ8GrTx I 1,007 ± 600 2,690 ± 460LWCKKRL YCQKWMWTCDEERKCCEGLVCR9GsMTx  260 ± 50 2,699 ± 790LWCKRIINMII/PaTXII*Contain 3 disulfide bridges in C1-C4, C2-C5 and C3-C6.
Amino acids and their abbreviations are listed in the following table.
Amino Acid3 Letter Code1 Letter CodeAlanineAlaAGlutamineGlnQLeucineLeuLSerineSerSArginineArgRGlutamicGluEAcid/GlutamateLysineLysKThreonineThrTAsparagineAsnNGlycineGlyGMethionineMetMTryptophanTrpWAsparticAspDAcid/AspartateHistidineHisHPhenylalaninePheFTyrosineTyrYCysteineCysCIsoleucineIleIProlineProPValineValV
WO 2012/004664 A2 also discloses a C-terminal modified Protoxin II analog (SEQ ID NO: 10):
This analog shows similar potency to Nav1.7 as unmodified Protoxin II, but decreased selectivity for Nav1.7 over Nav1.2 as compared to unmodified Protoxin II:
SEQIDIC50 Nav1.7IC50 Nav1.2Sequence*NO:Name(nm)(nm)YCQKWMWTCDSERKCCEGMVCRLWCK10Protoxin1  8 ± 1KKLW-NH2II-NH2 YCQKWMWTCDSERKCCEGMVCRLWCK 1Protoxin1105 + 20KKLWII*Contain 3 disulfide bridges in C1-C4, C2-C5 and C3-C6.See WO 2012/004664 A2, Tables 2 and 3. The loss of selectivity is undesirable.